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Abstract

Background

Single nucleotide polymorphisms are common in duplicated genes, causing functional
preservation, alteration or silencing. The Plasmodium falciparum genes var2csa and Pf332 are duplicated in the haploid genome of the HB3 parasite line. Whereas the molecular
function of Pf332 remains to be elucidated, VAR2CSA is known to be the main adhesin
in placental parasite sequestration. Sequence variations introduced upon duplication
of these genes provide discriminative possibilities to analyze allele-specific transcription
with a bearing towards understanding gene dosage impact on parasite biology.

Results

We demonstrate an approach combining real-time PCR allelic discrimination and discriminative
RNA-FISH to distinguish between highly similar gene copies in P. falciparum parasites. The duplicated var2csa variants are simultaneously transcribed, both on a population level and intriguingly
also in individual cells, with nuclear co-localization of the active genes and corresponding
transcripts. This indicates transcriptional functionality of duplicated genes, challenges
the dogma of mutually exclusive var gene transcription and suggests mechanisms behind antigenic variation, at least in
respect to the duplicated and highly similar var2csa genes.

Conclusions

Allelic discrimination assays have traditionally been applied to study zygosity in
diploid genomes. The assays presented here are instead successfully applied to the
identification and evaluation of transcriptional activity of duplicated genes in the
haploid genome of the P. falciparum parasite. Allelic discrimination and gene or transcript localization by FISH not only
provide insights into transcriptional regulation of genes such as the virulence associated
var genes, but also suggest that this sensitive and precise approach could be used for
further investigation of genome dynamics and gene regulation.

Background

Gene duplications, insertions, deletions and single nucleotide polymorphisms (SNPs)
are genetic modifications responsible for creation of variable gene families, and
contribute to genetic diversity and functional divergence [1]. In humans, gene duplications and deletions have been shown to occur genome wide,
thus creating a vast source of genetic variation [2,3]. Genetic alterations are also common throughout the Plasmodium falciparum genome, many of which have been shown to correlate with phenotype alterations of this
lethal malaria parasite [4-10]. SNPs are commonly introduced in genes upon amplification, causing functional preservation,
alteration or dysfunctional alteration/silencing by degenerative mutations of the
additional gene copy. To fully understand the impact a particular gene amplification
could have on the biology of an organism, it is of major interest to discriminate
the paralogs in order to determine the respective gene copy's functionality. The sequence
variations introduced upon duplications or evolutionary drift present tools that could
enable this discrimination [11].

There is a highly nonrandom distribution of genetic variability in terms of functional
classes in P. falciparum [5], with the greatest variation in genes coding for proteins associated with the infected
red blood cell (iRBC) membrane, which are known to interact with the host immune system
[12]. These include the family of P. falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, employed by the parasite to sequester
in the microvasculature of various organs in the human host. This family, encoded
by approximately 60 var genes per haploid parasite genome [13-16], presents the parasite with variable surface antigens that enhance the parasite's
chances of survival and evasion of the host immune response [17,18]. Earlier studies have indicated that expression of PfEMP1 is mutually exclusive [19,20]; several var genes can be transcribed during the ring stage, but in each parasite only one dominant
full-length mRNA is transcribed, translated into protein and displayed on the iRBC
surface at the mature trophozoite stage [19,21,22].

In pregnancy-associated malaria, parasites bind receptors on the maternal side of
the placenta [23], of which chondroitin sulphate A (CSA) is believed to be the main receptor [24,25]. This binding is accomplished using the PfEMP1 VAR2CSA as the main parasite ligand
[21]. The gene encoding VAR2CSA (var2csa) was recently identified as duplicated in the culture-adapted P. falciparum HB3 parasite line [26], originally cloned from the Honduras I/CDC strain in 1983 [27].

The var2csa gene is found in nearly all P. falciparum isolates [28], and has been suggested to have an ancient origin due to the existence of a var2csa ortholog in the genome of the chimpanzee malaria parasite P. reichenowi. The gene is unusually conserved compared to other members of the var gene family, with observed diversification associated with segmental gene recombination
and gene conversion events [26,28]. Sequence polymorphisms between different var2csa genes mainly group into segments of limited diversity, with a few basic sequence types
within each segment [29]. The two var2csa paralogs in the HB3 genome are highly similar in sequence, displaying a nucleotide
sequence identity of 89.6% between the complete genes (HB3 genome sequence locus PFHG_05046.1
and PFHG_05047.1 versus locus PFHG_05155.1) and 91.6% between exon 1 (PFHG_05046.1
versus PFHG_05155.1) [30], which encodes the external part of PfEMP1. The sequence differences are found in
all parts of the gene, often concentrated into segments, with SNPs of mixed nature
(non-synonymous, synonymous and intronic).

Also duplicated in the HB3 genome is the gene Pf332, which encodes a suggested surface-associated parasite protein [31]. Pf332 is the largest known malaria protein associated with the iRBC surface but
relatively little is known about its molecular function. The protein is suggested
to be involved in modulation of RBC rigidity as well as RBC adhesion [32-34] but further studies are needed to scrutinize its function. Parasites with duplicated
genes, where functionality is preserved, could potentially be used as tools to elucidate
functions of genes of interest. The var2csa and Pf332 gene copies in HB3 display slight differences in sequence, thereby providing means
of discrimination.

Here, we demonstrate a TaqMan real-time PCR assay in which SNPs provide the basis
for discrimination between highly similar paralogs in a haploid genome. Using the
HB3 parasite line and assays discriminative towards sequence-variable alleles of both
var2csa and Pf332, we show that the different alleles of both genes are readily picked up at the DNA
level, with other parasite strains (NF54, FCR3 and Dd2) serving as positive and/or
negative controls for the respective alleles. Performing the same analysis on reverse
transcribed mRNA, we also show that both paralogs of var2csa and Pf332 are transcribed by the HB3 parasite, signifying transcriptional functionality of the
genes. Furthermore, single cell analyses with both real-time PCR allelic discrimination
and RNA-fluorescent in situ hybridization (RNA-FISH) with allele-specific probes confirmed that both gene copies
of var2csa are not only transcribed by individual parasites but also co-localize in the great
majority of cells. Highly specific yet facile, the real-time PCR assay provides a
useful tool for the investigation of the impact of gene duplications on the biology
of P. falciparum. Together with localization of genes and corresponding transcripts using FISH, this
provides important insights into potential mechanisms regulating surface-expressed
antigens on RBCs infected with P. falciparum.

Results and discussion

Description of designed allelic discriminative var2csa and Pf332 assays

Two sets of allele-specific real-time primers and TaqMan MGB probes, targeting two
different alleles (those encoding the DBL2x and DBL4ε domains, respectively), were
designed based on the fully sequenced genomes of P. falciparum isolates HB3 and Dd2 [30], and FCR3 and 3D7 [35,36]. 3D7 was originally cloned from NF54 [37] and appears to be isogenic to its ancestor considering similarities in var gene sequences, as seen by us in this study as well as by others [21]. The assays were designed to enable detection and discrimination of different var2csa variants in FCR3, NF54 and Dd2 and the two var2csa paralogs in the HB3 genome (Figure 1a, b). Assay 1 (Figure 1a) was deliberately designed to not amplify var2csa of parasite strain Dd2. The allele 2-specific probe of this assay matches the Dd2
var2csa sequence perfectly, but due to mismatched annealing sites for both forward and reverse
primers, genomic DNA (gDNA) from Dd2 could be used as negative amplification and detection
control.

Figure 1.var2csa and Pf332 alleles in P. falciparum genomes and discriminative assay design. Alignments of var2csa and Pf332 gene sequences gathered from the fully assembled 3D7 genome and the partially assembled
FCR3, Dd2 and HB3 genomes used for allelic discriminative assays. Accession numbers
or genome sequence contigs with strain names within parentheses are presented as a
means of identification for all alleles. Assays were designed towards two different
parts of var2csa, (a) DBL2x and (b) DBL4ε, and (c) to exon 1 of Pf332. Designed real-time PCR primers are indicated in light blue and probes for allele
1/wild type in green and allele 2/mutant in red. Discriminative SNPs are marked with
asterisks.

One assay was similarly designed to identify different Pf332 variants in NF54, FCR3 and HB3. The discriminative probes were designed towards a
non-synonymous SNP altering amino acid 326 in the translated protein from a serine
(wild type) to a proline (mutant) - S326P (Figure 1c). This SNP within the recently identified exon 1 of the very large Pf332 gene [32] has so far only been identified in the HB3 parasite genome [7,8], preventing the use of any other parasite line as a positive control for the mutant
allele. Very little is known about the role of the Pf332 protein and there is no information
about the transcription or function of the altered protein. The assay depicted in
Figure 1c provides the means for determining the transcription of Pf332.

Allelic discrimination assay validation

Amplification efficiencies and allele identification specificities were analyzed using
gDNA dilutions originating from HB3, NF54, FCR3 and Dd2 parasites. Serial dilutions
of HB3 gDNA were employed for amplification efficiency determinations for all three
designed assays, yielding highly similar efficiencies within assay components (Additional
data file 1). The near identical amplification efficiencies detected therefore provide
unbiased amplification of the respective alleles. The specificity and sensitivity
of designed primer/probe combinations were analyzed for the var2csa alleles using known ratios of NF54 and FCR3 gDNA (known to harbor specific single
alleles). The results clearly display ratio-dependent signals using both a cycle threshold
(Ct) value approach (detected Ct values for each allele within the assays for each
gDNA ratio mixture; Figure 2a, b) as well as a total fluorescence emission approach (amount of detected fluorescence
from both probes adjusted for background, that is, post-read compensated with a pre-read
signal deduction; Figure 2c, d). Due to the lack of a positive control for one of the Pf332 alleles, we used HB3 gDNA for assay validation. For the same serial dilution of gDNA
as used for amplification efficiency calculations, three different amplification reactions
were performed. The reactions all contained the primers of the assay and either the
wild-type detection probe, the mutant-detection probe or a mixture of the two. As
shown in Figure 2e, the assay is highly specific for the different alleles, with allele frequencies
maintained even at low DNA concentrations.

Figure 2. Quality and performance assessment of allelic discrimination assays. (a, b) Shown are cycle thresholds (Ct) achieved using different ratios of NF54 and FCR3 gDNA
and discriminative assays for var2csa DBL2x (a) and DBL4ε (b). Filled squares represent amplification of allele 1 (detected
with FAM) and filled circles indicate amplification of allele 2 (detected with VIC),
with error bars representing standard deviations. (c, d) Total fluorescence emission, with background deducted, for mixes of NF54 and FCR3
gDNA using the same assays also demonstrates the specificity of var2csa probes. (e) Pf332 allele specificity and concentration dependency is shown using serial dilutions of
HB3 gDNA. Amplifications in the presence of only wild-type probe (y-axis), mutant
probe (x-axis) or a combination of both (middle) are shown. No template controls (NTC)
consistently showed negligible signals in all experiments.

Presence of var2csa and Pf332 alleles in various P. falciparum genomes

In order to confirm the number of gene copies predicted from the fully or partially
assembled NF54, FCR3, Dd2 and HB3 genomes, we performed relative copy number analyses
of the var2csa and Pf332 genes. The HB3 genome is supposed to contain two full-length var2csa genes and three additional var2csa DBL4ε sequences, as shown in Figure 1a, b. Our results indicate that HB3 harbors just the two copies of var2csa without the additional DBL4ε sequences (compared to single copies in other parasites;
Figure 3a, b), suggesting that the DBL4ε sequences are likely due to the partial assembly of the
present HB3 genome. One of the var2csa paralogs in HB3 is located on chromosome 12 [26] whereas the location of the second is unknown. In order to determine the chromosomal
location of the second var2csa copy, we performed pulsed field gel electrophoresis (PFGE) followed by Southern blotting
with var2csa-specific probes. This revealed the additional var2csa paralog in HB3 to be located on chromosome 1 (Figure 3c).

Figure 3. Copy numbers of var2csa and Pf332 alleles and allelic discrimination. (a, b, f) var2csa and Pf332 gene copy numbers in various parasite strains are shown relative to the NF54 strain,
with error bars representing the confidence interval (CI 95%). Two gene copies were
identified in all cases for the HB3 parasite, suggesting that the three additional
DBL4ε sequences are due to the partial assembly of this fully sequenced genome. (c) PFGE followed by Southern blotting revealed the second var2csa copy to be located on chromosome 1 in HB3. An ethidium bromide stained PFGE gel is
shown on the left with separated chromosomes from HB3, NF54 and the standards Hansenula wingei and Saccharomyces cerevisiae; selected chromosome sizes are indicated in megabase-pairs (Mb). The Southern blot
shown on the right revealed the var2csa-specific DNA probe to hybridize to chromosome
1 in HB3 (indicated with an arrow). (d, e) Discrimination of var2csa alleles in gDNA from the indicated parasites showed single allele frequency in NF54,
Dd2 (Allele 1) and FCR3 (Allele 2), and double alleles in HB3. (g) The same analysis on the S326P mutation in Pf332 revealed only the wild-type version in NF54, whereas HB3, with its dual copies, harbors
both the wild-type and mutant versions.

The presence of the sequence-variable alleles was subsequently analyzed using the
discriminative method described above. The results show the expected patterns, with
single alleles of var2csa DBL2x in NF54 and FCR3 and the presence of both allele types in HB3. The negative
parasite control (Dd2) showed no amplification of either allele, proving specific
var2csa amplification and detection (Figure 3d). The assay for the var2csa DBL4ε amplification showed identical results, apart from the correct amplification
of the expected and correctly primed copy in Dd2 (Figure 3e).

The copy number analysis of Pf332 confirmed the duplication of this gene in HB3 (Figure 3f) and these paralogs were similarly proven to be sequence variable (Figure 3g). This gene constitutes one of the largest in the P. falciparum genome (18.5 kb) but the number of identified SNPs is relatively low [8,38]. The SNP used here for the discrimination of the two Pf332 copies is unique for the HB3 parasite (among the isolates so far sequenced) and could
thus be used as a tool for identity determination since cross-contaminated P. falciparum strains are relatively common [39]. Taken together, the described modus operandi demonstrates the possibility to discriminate duplicated genes based on limited sequence
variation.

The duplicated var2csa and Pf332 alleles are transcriptionally active

The presence of alleles at the DNA level says nothing about their transcriptional
activity, since silent transcripts are potentially created upon amplification of genes.
Hence, var2csa transcripts in different parasite lines were analyzed using the same allele-discriminating
approach described above. As illustrated in Figure 4a, b, var2csa transcripts were detected in all three parasite lines, both before and after CSA selection.
Transcriptional activity was confirmed for both var2csa genes in HB3 and HB3CSA as well as the respective single alleles in NF54/NF54CSA and
FCR3/FCR3CSA (Figure 4a, b). Transcripts of both Pf332 copies were similarly present in the HB3 parasite (Figure 4c), signifying preserved functionality of all duplicated alleles, at least at the transcriptional
level. These results reflect transcriptional activity in large populations of parasites
but give no information about whether both gene copies of var2csa and Pf332 are expressed in single cells.

Figure 4.var2csa and Pf332 allele-specific transcriptional activity. (a, b) Transcriptional activity was confirmed for both var2csa allele types in HB3 and HB3CSA, and the single allele types of FCR3, FCR3CSA, NF54
and NF54CSA. (c) Both Pf332 copies in HB3 were also actively transcribed, demonstrating transcriptional functionality
in these duplicated genes. Controls with RNA reverse transcribed without addition
of reverse transcriptase (RT-) and exchange of template for ddH2O (NTC) were included in all experiments to prevent signals from gDNA or contaminations
from influencing the interpretation of the results.

Single mature trophozoites transcribe both var2csa gene copies

Single HB3CSA parasites were collected using micromanipulation and further analyzed
with a nested PCR/real-time PCR approach. Surprisingly, both allele types of var2csa were observed to be transcribed in individual parasites collected at 24 ± 4 h post-invasion
(p.i.; Figure 5a). Transcription of both allele types was readily detected in the majority of cells
analyzed, independent of the use of the reverse transcription priming procedure (Figure
5b). For some of the single cells only one of the alleles was detected, either signifying
a true difference in transcription pattern among parasites or possibly due to introduced
bias in the first PCR amplification. Re-sequencing of the real-time PCR amplified
products confirmed the allele calls achieved with the allelic discriminative approach,
and revealed var2csa sequences exclusively, thus further proving the accuracy and specificity of the assay
(data not shown).

Figure 5.var2csa allele transcriptional activity in individual HB3CSA parasites. Single cell transcription
from 11 individual HB3 parasites repeatedly selected for CSA-binding phenotype. Parasites
at the mature trophozoite stage (24 ± 4 h p.i.) were subjected to three different
priming strategies during the reverse transcription (random primers and oligo(dT),
oligo(dT) only and var2csa-specific primers). (a) Allele frequencies of alleles 1 and 2 for the positive gDNA controls NF54 (filled
circles), FCR3 (filled triangles) and HB3 (filled squares) as well as for the 11 cDNA
samples (empty square). Negative controls (filled diamonds) with RNA reverse transcribed
without addition of reverse transcriptase (RT-), exchange of template for ddH2O (NTC) and amplifications from uninfected red blood cells (RBCs) were included in
all experiments to prevent signals from gDNA, contaminations or unspecific amplifications
influencing the interpretation of the results. All data-points represent means of
triplicates with standard deviations for each allele expressed as bi-directional error
bars. (b) Mean allele frequencies and predicted allele calls for all samples and priming strategies
described above. N.D., not detected.

RNA-FISH was used to visualize and confirm the results from the single cell real-time
PCR assays. RNA probes were designed towards one of the most sequence-variable regions
of the two var2csa paralogs (towards the 5' end) in order to discriminate between them (Figure 6a). The area chosen also presented the possibility of using NF54CSA and FCR3CSA as
controls for one of the sequence types in this particular region of var2csa. Most HB3CSA parasites (16 ± 4 h p.i.) were indeed shown to have a high abundance
of transcripts from both var2csa paralogs, whereas NF54CSA and FCR3CSA displayed only transcripts from their single
allele types (Figure 6b, c). Control probes towards antisense transcripts of var2csa consistently showed no hybridization (data not shown), which is expected due to previous
findings of only low levels in CSA-selected FCR3 parasites [40] and a preponderance of var gene antisense transcripts appearing at later stages and then mostly limited to the
3' end of exon 1 [41]. In addition, probes generated towards the kahrp gene were included as positive controls [42] and hybridized in expected patterns in all three parasites (Figure 6d).

Figure 6.Detection of transcripts from both var2csa paralogs in P. falciparum parasites as seen by RNA-FISH. (a) Alignment of variable var2csa sequences serving as template for RNA probes (PFHG_05046.1 and PFHG05155.1) designed
to discriminate between the two var2csa alleles in HB3CSA (asterisks indicate variable nucleotides between the two alleles
in HB3CSA). The sequences of FCR3CSA and NF54CSA display high homology to PFHG_05155.1
in this particular region of the var2csa gene, with limited variability (denoted by arrows) allowing detection of the single
copies in these parasites at the selected FISH stringency. Percent identical nucleotides
between the sequences are displayed (with PFHG_05155.1 as 100%) to the bottom right.
(b) Representative pictures of the hybridization patterns achieved with the two probes
targeting var2csa mRNA in indicated parasites. The probe generated from PFHG_05155.1 is displayed in
green; the probe towards PFHG_05046.1 in red and parasite nuclei stained with DAPI
in blue. Probes towards antisense transcripts consistently showed no hybridization
(data not shown). (c) Pictures representing the three scenarios observed for detection of var2csa allele transcripts in HB3CSA, with frequencies for each scenario (transcripts detected
from both paralogs, transcripts detected from only allele 1, and transcripts detected
from only allele 2) given as a percentage in each representative picture (n = 92).
The great majority of simultaneously transcribed duplicated var2csa genes in single HB3CSA cells were exclusively accompanied by the observation of co-localization
of the two transcripts in the nuclei. (d) Representative hybridization patterns achieved with the control probe targeting the
kahrp gene (red) in nuclei (blue) of all the CSA-selected parasite lines used. For the var2csa hybridizations, the negative control probes towards antisense transcripts of kahrp revealed no detection of hybridization.

Apart from confirming simultaneous transcription of the two var2csa paralogs in single HB3CSA cells, the RNA-FISH analysis also revealed an intriguing
exclusive nuclear co-localization of the two transcripts (Figure 6b, c), this despite the genes being localized on different chromosomes (see above). DNA-FISH,
performed in order to further investigate var2csa localization in the nucleus of HB3CSA parasites, revealed that the genes also co-localize
in the majority of cells (Figure 7, scenarios I and II). The extent of co-localization (78.5%) corresponded very well
with the fraction of cells transcribing both paralogs as seen with real-time PCR and
RNA-FISH. Previous studies have contradictorily suggested that var genes are either distant from and/or adjacent to telomeric ends when active [22,43-46]. Here, the var2csa genes in HB3CSA appear both to be distant from and co-localize with Rep20 (representing
telomeric clusters), although the latter is more common (Figure 7b).

Figure 7.Subnuclear localization of the duplicated var2csa genes in HB3CSA parasites. Representative DNA FISH pictures illustrating the five
different localization patterns of both var2csa paralogs (green), telomeric clusters (rep20, red) in nuclei stained with DAPI (blue).
I, co-localized var2csa alleles where both also co-localized with rep20; II, co-localized var2csa alleles that did not co-localize with rep20; III, non-co-localized var2csa alleles but both co-localized with rep20; IV, non-co-localized var2csa alleles with neither allele co-localized with rep20; V, non co-localized var2csa alleles with one allele co-localized with rep20. The var2csa paralogs were exclusively found towards the rim of the nuclei. (b) Quantification of the described localization patterns illustrated in a pie-chart with
percentages (n = 204). Co-localized var2csa paralogs (I and II) are shaded with stripes and constitute 78.5% of the total.

The transcription of var genes at the mature trophozoite stage is presumed to be mutually exclusive [19,20]. However, as shown here, both var2csa copies of HB3CSA are simultaneously active in individual cells, challenging the dogma
of mutually exclusive transcription of var gene family members. Previous studies have indicated that CSA-selected parasites transcribe
more than one var gene at the mature trophozoite stage [47]. The results of this study are intriguing, especially since both alleles were detected
using only oligo(dT) primers in the reverse transcription, suggesting that the transcripts
were destined for translation. This was also supported by the common observation of
the cytoplasmic localization of both var2csa transcripts in cells transcribing both paralogs using RNA-FISH. This is markedly different
from the case of var1csa (varCOMMON), which is suggested to be simultaneously transcribed along with other var genes at the mature trophozoite stage, but to only produce sterile transcripts [48]. The interesting observation of co-localized native var2csa genes and transcripts argues for a previously suggested active site of var gene transcription [43,45,46]. Whether genes reposition from telomeric clusters within the heterochromatin region
into the euchromatin portion of the nuclear periphery upon activation has been both
supported [22,43,49] and debated against [44,50]. In the HB3CSA parasites studied here, repositioning (as viewed with Rep20) did not
seem necessary for transcriptional activity of the duplicated var2csa genes, something that has been shown previously for var2csa [22,49]. Despite this controversy, all studies conducted on nuclear localization are consistent
with the existence of a specific var gene expression site that is apparently able to accommodate more than one active var gene, as shown here as well as by Dzikowski et al. [45,46], and is perhaps determined by activating and repressive histone methyl modifications
[51] rather than by gene position in relation to telomeric clusters.

Even though it challenges the dogma of mutually exclusive var gene transcription, simultaneously active duplicated var2csa genes could be a special case, since the sequence similarity among different var2csa variants is high compared to that of other var genes. The fact that one var2csa allele in HB3 is located in the subtelomeric region of chromosome 12 [26] whereas the second allele is located on chromosome 1 (Figure 3c) argues that the transcription of var2csa is regulated by trans-acting factors rather than cis-acting elements. This could suggest the presence of var2csa-specific transcription factors with preserved DNA-binding regions in the duplicated
gene copies. The upstream regions of the var2csa paralogs are indeed highly similar (data not shown, but available at Broad Institute
of Harvard and MIT [30]), so both are likely bound by analogous DNA binding trans-acting elements, thereby
enabling their co-transcription, a so far poorly evaluated mechanism of antigenic
variation in P. falciparum. However, even though transcriptional activity of both genes is shown here, with
transcripts suggested to undergo translation, the function(s) of the sequence polymorphic
proteins is not known. Whether both transcripts are indeed translated [7] and proteins exported to the surface of the parasitized erythrocyte and whether functionality
is preserved or altered remain to be elucidated in order to understand the impact
of this gene duplication on the development of pregnancy-associated malaria.

Conclusions

Using allele discriminating real-time PCR assays in conjunction with RNA-FISH, with
SNPs providing the basis for distinction, we have identified duplicated but slightly
sequence variable gene copies in haploid genomes of P. falciparum. The different alleles were also proven to be transcriptionally active, an important
finding with regard to determining the functionality of duplicated genes. This is
the first report in malaria research where allele-specific probes have been used not
only to distinguish gene variants and sequence variable gene copies at the genomic
level, but also to accurately discriminate allele-specific transcripts. The possibility
to differentiate transcripts of the var2csa paralogs with the two different methodologies not only showed transcription of two
var gene copies in single mature trophozoite stage parasites, but also that these co-localized
in a great majority of cell nuclei. Not only do these findings challenge the dogma
of mutually exclusive var gene transcription, they also add complexity to the understanding of the molecular
basis of antigenic variation and virulence in pregnancy-associated malaria. The approach
can be extended to study other issues related to genetic polymorphisms in malaria
- for example, tp determine whether the transcription of members of gene families
other than the var family is mutually exclusive. Highly specific yet facile and time efficient, this
allelic discrimination assay provides a useful tool for the investigation of the impact
of gene duplications on the biology of P. falciparum as well as mechanisms regulating surface-expressed antigens on red blood cells infected
with P. falciparum. A more thorough insight into the field of genetic differences and the mechanisms
behind these could generate a better understanding of the biology of P. falciparum, as well as of the molecular aspects of the pathogenesis of malaria.

Materials and methods

Parasite cultivation and CSA-selection

Parasites were maintained in continuous culture according to standard procedures [52]. Parasite lines selected for CSA binding phenotype were repeatedly panned on CSA-coated
plastic; 10 μg/ml CSA in phosphate-buffered saline (PBS) was coated on non-tissue
culture treated six-well plates overnight in a humid chamber at 4°C. 2% bovine serum
albumin in PBS was added for 30 minutes in order to block non-specific binding. Mid-late
stage trophozoites were purified using a MACS magnetic cell sorter (Miltenyi BioTec,
Bergisch Gladbach, Germany). Purified iRBCs (80 to 90% parasitemia) were washed in
RPMI-1640, resuspended in malaria culture medium with 10% human serum and added to
CSA-coated wells (approximately 108 iRBCs per well). Plates were incubated according to standard parasite cultivating
procedures for 1 h at 37°C with gentle rocking every 15 minutes. Wells were then washed
with malaria culture medium until background binding was low. Finally, 2 ml malaria
culture medium with 10% human serum and 100 μl fresh blood was added to each well
and plates incubated at 37°C. Parasite suspensions were moved to culture flasks after
24 h and used in downstream experiments. CSA-binding assays were performed according
to standard procedures [24].

Nucleic acid extraction

gDNA was prepared using Easy-DNA Kit (Invitrogen, California, USA) following the supplier's
recommendations with minor modifications. The gDNA-containing aqueous phase, once
extracted with 25:24:1 phenol:chloroform:isoamyl alcohol (Sigma, St. Louis, Missouri,
USA) was RNase treated before one additional round of extraction. Total RNA was harvested
at 16 h p.i. for var2csa assays and 24 h p.i for Pf332 assays using an RNeasy Mini Kit (Qiagen, California, USA). Samples were DNase treated
in order to remove contaminating gDNA (Turbo DNase, Ambion, Texas, USA) and reverse
transcribed (Superscript III RNase H reverse transcriptase, Invitrogen). For each
cDNA synthesis reaction, a control reaction without reverse transcriptase (no template
control (NTC)) was performed with identical amounts of template.

Pulsed field gel electrophoresis and Southern blotting

PFGE using the CHEF Mapper system and pulse field certified agarose (Bio-Rad, California,
USA) was performed in order to fractionate HB3 and 3D7 chromosomes. Chromosomes 1
to 4 were separated on a 1% gel in 0.5× TBE buffer with switch times ramped from 60
to 120 s at 6 V cm-1 in a 120° pulse angle for 28 h at 14°C. Chromosomes 5 to 10 were separated on a 1%
gel in 0.5× TBE buffer at 14°C with a switch time of 120 s at 4.5 V cm-1 in 120° pulse angle for 22 h followed by a 240 s switch time for 33 h. Chromosomes
11 to 14 were separated on a 0.4% gel in 0.5× TBE buffer with switch times ramped
from 120 to 720 s at 2.5 V cm-1 in a 120° pulse angle for 66 h at 18°C. DNA was then transferred to Hybond N+ nylon
membranes (GE Healthcare, Stockholm, Sweden) using standard procedures. Southern blotting
was performed using an approximately 1-kb double-stranded DNA probe targeting both
var2csa alleles of HB3, using the primer pair var2csa lr1 described in Table 1. In brief, target sequences were amplified in a standard PCR reaction using HB3 gDNA
as template followed by a second PCR reaction using the first PCR product as template.
The final products were separated on an agarose gel, excised and gel extracted using
the QIAquick Gel Extraction Kit (Qiagen), labeled with DIG using DIG High Prime Kit
(Roche Applied Science, Indianapolis, USA) and purified using a QIAquick PCR Purification
Kit (Qiagen), following the manufacturer's recommendations. Nylon membranes were incubated
with 25 ng/ml DIG-labeled probe at 42°C with gentle agitation overnight. The membranes
were subsequently washed under high stringency conditions under agitation (2× SSC,
0.1% SDS for 2 × 5 minutes at room temperature followed by 0.5× SSC, 0.1% SDS for
2 × 15 minutes at 68°C) before detection using CSPD (Roche Applied Science) following
the recommendations of the manufacturer.

Design of real-time PCR allelic discrimination primers and probes

The fully sequenced genomes of FCR3, Dd2 and HB3 and the partly assembled genome of
3D7 were used as templates for the allelic discrimination assays. Seed sequences from
the 3D7 var2csa (PFL0030c) and Pf332 (PF11_0506) genes [36] were blasted (WashU BLASTN on the BLOSUM62 matrix without low complexity filter)
towards the HB3 and Dd2 genomes [30] and the FCR3 genome [35]. Retrieved sequences were analyzed for areas containing moderate sequence variability
suitable for conserved primer annealing sites and sequence variable and discriminative
probe annealing sites. Primers and probes (MGB probes labeled with either FAM or VIC)
for suitable genome contigs were manually designed using Primer Express 3.0 (Applied
Biosystems, California, USA). Great care was taken to ensure a high theoretical discrimination
possibility, a low risk of primer dimerization and secondary structure formation of
all primers and probes (assessed using ΔG estimations in NetPrimer, Premier Biosoft,
Palo Alto, California, USA). Specificities of designed assays were confirmed through
blasting towards all genomes used as templates.

Relative gene copy number determination

Relative copy number determinations were performed in quadruplicate in MicroAmp 96-well
plates (Applied Biosystems, California, USA) in 20 μl, containing Power SYBR green
master mix and primers towards var2csa (300 nM of forward and reverse primers of primer pair 1 and 600 nM of forward and
reverse primers of primer pair 2), Pf332 (300 nM of both forward and reverse primers) and 2 ng of template. As endogenous controls
(assumed to exist as single copy genes in every genome) we employed primers for β-tubulin (300 nM of forward and reverse primers) and seryl-tRNA synthetase (900 nM of forward and reverse primers). Concentrations of primers for the target
and reference genes were optimized using serially diluted NF54 gDNA and amplification
efficiencies of primers using stated concentrations were sufficiently close to obviate
the need for a correction factor. Amplification reactions were carried out in an ABI
7500 real-time PCR system in 40 cycles (95°C for 15 s, and 60°C for 1 minute). Data
were analyzed using the Applied Biosystems 7500 system software version 1.3.1 and
relative copy numbers were computed according to the ΔΔCt method using a statistical
confidence interval of 95%.

Allelic discrimination

Performance of allelic discrimination assays was also evaluated prior to conducting
allele discrimination experiments. Amplification efficiencies of all assays and the
allele discrimination capability the Pf332 assay were determined using serial dilutions of HB3 gDNA. PCR reactions were performed
in quadruplicate in MicroAmp 96-well plates in 20 μl, containing TaqMan buffer with
UNG (Applied Biosystems), 900 nm of each forward and reverse primer and 200 nM of
either probe. Amplifications were conducted in an ABI 7500 real-time PCR system, starting
with a pre-read (for background fluorescence measurement) followed by 40 cycles of
amplification (95°C for 15 s, and 60°C for 1 minute) and a final post-read (for total
fluorescence emission measurement after amplification). Standard curves for efficiency
determination were plotted after the detection threshold was set above the mean baseline
value for the first 3 to 15 cycles. Efficiencies of amplification and detection of
respective alleles within all assays were shown to be close to identical, proving
unbiased allele recognition. Obtained post-read data (adjusted for the background
detected in the pre-read) from amplifications of Pf332 alleles using the wild-type-detecting FAM-labeled probe, the mutant-detecting VIC-labeled
probe and a combination of both probes were used to assess proper allele frequency
detection. Proper allele frequency detection was similarly determined for the two
var2csa allelic discrimination assays using gDNA from NF54 and FCR3 in various mixes. Allele
detection frequencies were analyzed using both cycle thresholds for the amplification
(with standard deviations expressed as error bars) as well as post-read data. Allelic
discrimination experiments were subsequently performed using validated assays on gDNA
from NF54, FCR3, Dd2 and HB3 and cDNA from NF54, FCR3 and HB3. Approximately 2 ng
of template, the above described primer and probe concentrations, TaqMan buffer with
UNG and the same amplification scheme as described above were used. All experiments
included NTCs, with ddH2O used instead of gDNA for the analysis of the presence of alleles in genomes and
reverse transcription control reactions lacking reverse transcriptase for the analysis
of allele-specific transcription.

Single cell nested allelic discrimination

Single iRBCs were picked according to the previously described methodology [19]. In brief, micromanipulation was conducted on highly synchronous HB3CSA cultures
(24 ± 4 h p.i.) using a micromanipulator MN-188 (Narishige, Tokyo, Japan), sterile
micropipettes (approximately 3 μm internal diameter) and an inverted microscope (Nikon
Diaphot 300). A total of 11 iRBCs and 4 RBCs (picked as control) were collected in
this manner. Picked cells were deposited in 9 μl drops of 1× Superscript III buffer
(Invitrogen) containing 1:30 RNase inhibitor (SUPERase-In, Invitrogen) on multiwell
slides pre-treated with dichlorodimethylsilane, and transferred to PCR tubes. Cells
were immediately frozen on dry ice before being heated to 94°C for 3 minutes and subsequently
DNase treated at 37°C for 30 minutes (rDNase 1, Applied Biosystems, California, USA).
Reverse transcription (Superscript III RNase H reverse transcriptase, Invitrogen),
was performed according to the manufacturer's recommendations at 50°C for 2 h using
three different priming approaches: random primers and oligo(dT)12-18 (Invitrogen), oligo(dT)12-18 only, or var2csa-specific primers (Table 1). For each cDNA synthesis reaction, a control reaction without reverse transcriptase
(RT-) was performed. We applied a nested PCR approach, initially amplifying an approximately
1-kb fragment of var2csa DBL2x with primers (Table 1, var2csa nest 1) external to the allele discriminative DBL2x assay. PCR reactions were performed
using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) with initial denaturation
at 95°C for 5 minutes followed by 35 cycles of amplification (95°C for 30 s, 58°C
for 45 s, 68°C for 1 minute 20 s) and final extension at 72°C for 7 minutes. Thereafter,
these amplicons were used as templates in real-time PCR allelic discrimination reactions,
using primers and probes as described above.

Fluorescent in situ hybridization

RNA-FISH was performed in order to visualize simultaneous transcription of both var2csa paralogs in single cells of HB3CSA. Single-stranded antisense and sense RNA probes
(Figure 6a) were generated towards one of the most variable regions of the two paralogs using
var2csa allele-specific primers with Sp6 promoter tails (Table 1) and subsequent in vitro transcription. Probes targeting the kahrp gene (used as control) were generated using the same procedure. Probes were transcribed
in the presence of either fluorescein or biotin and Sp6 RNA polymerase (Roche Applied
Science) according to the manufacturer's recommendations. Labeled probes were Dnase
treated and purified on Sephadex G-50 fine columns (GE Healthcare, Stockholm, Sweden)
and stored at -70°C until use. Highly synchronous HB3CSA, FCR3CSA and NF54CSA parasites
(16 ± 4 h p.i.) were isolated from their host erythrocytes using saponin (0.05% w/v)
and deposited as monolayers on Denhardt's coated microscope slides. Monolayers were
quickly air-dried, immediately fixed in ice-cold 100% ethanol for 2 minutes and subsequently
rehydrated through a series of ice cold 90%, 70% and 50% ethanol for 2 minutes each.
Slides were further fixed using 4% paraformaldehyde in PBS for 10 minutes at 4°C followed
by wash in 2× SSPE (3.0 M Sodium Chloride, 0.2 M Sodium Hydrogen Phosphate, 0.02 M
EDTA, pH 7.4) for 3 × 2 minutes. To permeabilize cells, slides were incubated in 0.1
M Tris-HCl with 0.01 M EDTA (pH 8.0) containing 5 μg/ml proteinase K for 10 minutes
at room temperature followed by washes in 2× SSPE for 3 × 2 minutes. In order to strip
basic proteins from the cells, slides were immersed in 0.2 M HCl for 15 minutes at
room temperature followed by washes in 2× SSPE for 3 × 2 minutes. Monolayers were
allowed to equilibrate/block in hybridization buffer without probe (10% dextran sulphate,
5× Denhardt's, 50% formamide, 2× SSC, 0.5% SDS, 1:100 RNA protector, 20 μl of 200
mg/ml yeast tRNA in Diethylpyrocarbonate (DEPC) H2O). RNA probes were denatured at 65°C for 5 minutes and thereafter immediately put
on ice before being resuspended in pre-heated hybridization buffer. Probe mix was
then added to slides, which were subsequently covered with a plastic cover and incubated
overnight at 42°C. Slides were then washed with 2× SSPE/50% formamide for 15 minutes
at 42°C, 2× SSPE for 15 minutes at 42°C, 0.2× SSPE for 15 minutes at 42°C and 0.2×
SSPE for 15 minutes at room temperature. Slides were blocked in 1× maelic acid/1×
blocking buffer (DIG wash and block buffer set, Roche Applied Science) supplemented
with 0.15 M NaCl for 30 minutes at room temperature. Biotin labeled probes were detected
with Neutravidin-Texas Red (Molecular Probes, California, USA) diluted 1:500 in 1×
maelic acid/1× blocking buffer/0.15 M NaCl for 30 minutes at room temperature. Slides
were thereafter washed tice in 1× maelic acid/1× blocking buffer/0.15 M NaCl for 10
minutes at room temperature followed by washing in 1× maelic acid/0.15 M NaCl/0.5%
Tween 20 for 2 × 10 minutes at room temperature. Preparations were mounted in Vectashield
containing DAPI (Vector Laboratories, California, USA), visualized using a Leica DMRE
microscope and imaged with a Hamamatsu C4880 cooled CCD camera.

DNA-FISH on HB3CSA (16 ± 4 h p.i.) was conducted according to the previously described
methodology [22], using primers var2csa lr1 and lr2 (Table 1) to generate var2csa probes recognizing both var2csa paralogs in HB3. Synthesis of these probes was performed as described above for Southern
blot probes, but labeled with fluorescein using a Fluorescein High Prime Kit (Roche
Applied Science) following the instructions of the supplier. Rep20, in a linearized
puc9 plasmid, was similarly labeled with biotin using the Biotin High Prime Kit and
used for co-localization of the var2csa genes with chromosomal telomere ends. Preparations were mounted in Vectashield containing
DAPI (Vector Laboratories), visualized using a Leica DMRE microscope and imaged with
a Hamamatsu C4880 cooled CCD camera. The physical localization of the two var2csa paralogs was determined and quantified for the frequency: co-localized var2csa alleles that also co-localized with rep20; co-localized var2csa alleles but with no co-localization with rep20; non co-localized var2csa alleles where both co-localized with rep20; non-co-localized var2csa alleles where neither allele co-localized with rep20; and non-co-localized var2csa alleles with only one of the alleles co-localizing with rep20.

Authors' contributions

UR, KB and QC conceived and designed the experiments. KB, UR, SN and JA performed
the experiments. KB, UR, KM and QC analyzed the data. QC and MW contributed reagents/materials/analysis
tools. KB, UR and QC wrote the manuscript. All authors read and approved the final
manuscript.

Additional data files

The following additional data are available with the online version of this paper:
a figure showing amplification efficiencies of allelic discrimination assays (Additional
data file 1).

Additional data file 1. Graphs showing standard curves of amplifications using primer pairs towards (a) var2csa DBL2X, (b) var2csa DBL4ε and (c) Pf332 S326P together with detection by allele-specific FAM- or VIC-labeled probes. Serial
dilutions of HB3 gDNA were used in all reactions. Filled squares show amplification
detected with FAM-labeled probe and filled circles detection with VIC-labeled probe.
Amplification efficiencies for primers and probes within all respective allele assays
were sufficiently close to obviate the need for a correction factor.